Treatment of tumor protein 63 (p63) related diseases by inhibition of natural antisense transcript to p63

ABSTRACT

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Tumor Protein 63 (p63), in particular, by targeting natural antisense polynucleotides of Tumor Protein 63 (p63). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of p63.

The present application is a divisional of U.S. application Ser. No.13/519,477 filed on Jun. 27, 2012, now U.S. Pat. No. 8,962,585, which isa National Stage Entry of International Application No.PCT/US2010/062326 filed on Dec. 29, 2010, which claims priority to U.S.Provisional Application No. 61/290,540 filed on Dec. 29, 2009, which areall incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

Embodiments of the invention comprise oligonucleotides modulatingexpression and/or function of p63 and associated molecules.

BACKGROUND

DNA-RNA and RNA-RNA hybridization are important to many aspects ofnucleic acid function including DNA replication, transcription, andtranslation. Hybridization is also central to a variety of technologiesthat either detect a particular nucleic acid or alter its expression.Antisense nucleotides, for example, disrupt gene expression byhybridizing to target RNA, thereby interfering with RNA splicing,transcription, translation, and replication. Antisense DNA has the addedfeature that DNA-RNA hybrids serve as a substrate for digestion byribonuclease H, an activity that is present in most cell types.Antisense molecules can be delivered into cells, as is the case foroligodeoxynucleotides (ODNs), or they can be expressed from endogenousgenes as RNA molecules. The FDA recently approved an antisense drug,VITRAVENE™ (for treatment of cytomegalovirus retinitis), reflecting thatantisense has therapeutic utility.

SUMMARY

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

In one embodiment, the invention provides methods for inhibiting theaction of a natural antisense transcript by using antisenseoligonucleotide(s) targeted to any region of the natural antisensetranscript resulting in up-regulation of the corresponding sense gene.It is also contemplated herein that inhibition of the natural antisensetranscript can be achieved by siRNA, ribozymes and small molecules,which are considered to be within the scope of the present invention.

One embodiment provides a method of modulating function and/orexpression of an p63 polynucleotide in patient cells or tissues in vivoor in vitro comprising contacting said cells or tissues with anantisense oligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to a reversecomplement of a polynucleotide comprising 5 to 30 consecutivenucleotides within nucleotides 1 to 288 of SEQ ID NO: 2 therebymodulating function and/or expression of the p63 polynucleotide inpatient mils or tissues in vivo or in vitro.

In an embodiment, an oligonucleotide targets a natural antisensesequence of p63 polynucleotides, for example, nucleotides set forth inSEQ ID NOS: 2, and any variants, alleles, homologs, mutants,derivatives, fragments and complementary sequences thereto. Examples ofantisense oligonucleotides are set forth as SEQ ID NOS: 3 to 6.

Another embodiment provides a method of modulating function and/orexpression of an p63 polynucleotide in patient cells or tissues in vivoor in vitro comprising contacting said cells or tissues with anantisense oligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to a reversecomplement of the an antisense of the p63 polynucleotide; therebymodulating function and/or expression of the p63 polynucleotide inpatient cells or tissues in vivo or in vitro.

Another embodiment provides a method of modulating function and/orexpression of an p63 polynucleotide patient cells or tissues in vivo orin vitro comprising contacting said cells or tissues with an antisenseoligonucleotide 5 to 30 nucleotides in length wherein saidoligonucleotide has at least 50% sequence identity to an antisenseoligonucleotide to an p63 antisense polynucleotide; thereby modulatingfunction and/or expression of the p63 polynucleotide patient cells ortissues in vivo or in vitro.

In an embodiment, a composition comprises one or more antisenseoligonucleotides which bind to sense and/or antisense p63polynucleotides.

In an embodiment, the oligonucleotides comprise one or more modified orsubstituted nucleotides.

In an embodiment, the oligonucleotides comprise one or more modifiedbonds.

In yet another embodiment, the modified nucleotides comprise modifiedbases comprising phosphorothioate, methylphosphonate, peptide nucleicacids, 2′-O-methyl, fluoro- or carbon, methylene or other locked nucleicacid (LNA) molecules. Preferably, the modified nucleotides are lockednucleic acid molecules, including α-L-LNA.

In an embodiment, the oligonucleotides are administered to a patientsubcutaneously, intramuscularly, intravenously, or intraperitoneally.

In an embodiment, the oligonucleotides are administered in apharmaceutical composition. A treatment regimen comprises administeringthe antisense compounds at least once to patient; however, thistreatment can be modified to include multiple doses over a period oftime. The treatment can be combined with one or more other types oftherapies.

In an embodiment, the oligonucleotides are encapsulated in a liposome orattached to a carrier molecule (e.g. cholesterol, TAT peptide).

Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of real time PCR results showing the foldchange+standard deviation in p63 mRNA after treatment of HEPG2 cellswith phosphorothioate oligonucleotides introduced using Lipofectamine2000, as compared to control. Real time PCR results show that the levelsof p63 mRNA in HEPG2 cells are significantly increased 48 h aftertreatment with one of the oligos designed to p63 antisense. Bars denotedas CUR-1172, CUR-1173, CUR-1174, CUR-1175 correspond to samples treatedwith SEQ ID NOS 3, 4, 5 and 6 respectively.

Sequence Listing Description—SEQ ID NO: 1: Homo sapiens tumor proteinp63 (Tp63), transcript variant 3, mRNA, mRNA, (NCBI Accession No.:NM_001114979); SEQ ID NO: 2: Natural p63 antisense sequence Hs.553263;SEQ ID NOs: 3 to 6—Antisense oligonucleotides. * indicatesphosphothioate bond.

DETAILED DESCRIPTION

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. The present invention is not limited by the ordering ofacts or events, as some acts may occur in different orders and/orconcurrently with other acts or events. Furthermore, not all illustratedacts or events are required to implement a methodology in accordancewith the present invention.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inan embodiment, the genes or nucleic acid sequences are human.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” ran mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, the term “mRNA” means the presently known mRNAtranscript(s) of a targeted gene, and any flintier transcripts which maybe elucidated.

By “antisense oligonucleotides” or “antisense compound” is meant an RNAor DNA molecule that binds to another RNA or DNA (target RNA, DNA). Forexample, if it is an RNA oligonucleotide it binds to another RNA targetby means of RNA-RNA interactions and alters the activity of the targetRNA. An antisense oligonucleotide can upregulate or downregulateexpression and/or function of a particular polynucleotide. Thedefinition is meant to include any foreign RNA or DNA molecule which isuseful from a therapeutic, diagnostic, or other viewpoint. Suchmolecules include, for example, antisense RNA or DNA molecules,interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA,enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA,antisense oligomeric compounds, antisense oligonucleotides, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds that hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, partiallysingle-stranded, or circular oligomeric compounds.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. The term “oligonucleotide”, alsoincludes linear or circular oligomers of natural and/or modifiedmonomers or linkages, including deoxyribonucleosides, ribonucleosides,substituted and alpha-anomeric forms thereof, peptide nucleic acids(PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate,and the like. Oligonucleotides are capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, Hoögsteen orreverse Hoögsteen types of base pairing, or the like.

The oligonucleotide may be “chimeric”, that is, composed of differentregions. In the context of this invention “chimeric” compounds areoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one monomer unit, i.e., a nucleotide inthe case of an oligonucleotides compound. These oligonucleotidestypically comprise at least one region wherein the oligonucleotide ismodified in order to exhibit one or more desired properties. The desiredproperties of the oligonucleotide include, but are not limited, forexample, to increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. Different regions of the oligonucleotide may thereforehave different properties. The chimeric oligonucleotides of the presentinvention can be formed as mixed structures of two or moreoligonucleotides, modified oligonucleotides, oligonucleosides and/oroligonucleotide analogs as described above.

The oligonucleotide can be composed of regions that can be linked in“register”, that is, when the monomers are linked consecutively, as innative DNA, or linked via spacers. The spacers are intended toconstitute a covalent “bridge” between the regions and have in preferredcases a length not exceeding about 100 carbon atoms. The spacers maycarry different functionalities, for example, having positive ornegative charge, carry special nucleic acid binding properties(intercalators, groove binders, toxins, fluorophors etc.), beinglipophilic, inducing special secondary structures like, for example,alanine containing peptides that induce alpha-helices.

As used herein “p63” and “Tumor Protein 63” are inclusive of all familymembers, mutants, alleles, fragments, species, coding and noncodingsequences, sense and antisense polynucleotide strands, etc.

As used herein, the words Tumor protein 63, Tumor protein 63,Transformation-related protein 63, p63, p63, Tp63, AIS, B(p51A),B(p51B), Chronic ulcerative stomatitis protein, CUSP, EEC3, Keratinocytetranscription factor KET, KET, LMS, NBP, OFC8, p40, p51, p53CP, p73H,P73H, p73L, P73L, RHS, SHFM4, TP53CP, TP53L, TP73L and Tumor proteinp73-like, are considered the same in the literature and are usedinterchangeably in the present application.

As used herein, the term “oligonucleotide specific for” or“oligonucleotide which targets” refers to an oligonucleotide having asequence (i) capable of forming a stable complex with a portion of thetargeted gene, or (ii) capable of forming a stable duplex with a portionof a mRNA transcript of the targeted gene. Stability of the complexesand duplexes can be determined by theoretical calculations and/or invitro assays. Exemplary assays for determining stability ofhybridization complexes and duplexes are described in the Examplesbelow.

As used herein, the term “target nucleic acid” encompasses DNA, RNA(comprising premRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA, coding, noncoding sequences, sense or antisensepolynucleotides. The specific hybridization of an oligomeric compoundwith its target nucleic acid interferes with the normal function of thenucleic acid. This modulation of function of a target nucleic acid bycompounds, which specifically hybridize to it, is generally referred toas “antisense”. The functions of DNA to be interfered include, forexample, replication and transcription. The functions of RNA to beinterfered, include all vital functions such as, for example,translocation or the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of an encoded product oroligonucleotides.

RNA interference “RNAi” is mediated by double stranded RNA (dsRNA)molecules that have sequence-specific homology to their “target” nucleicacid sequences. In certain embodiments of the present invention, themediators are 5-25 nucleotide “small interfering” RNA duplexes (siRNAs).The siRNAs are derived from the processing of dsRNA by an RNase enzymeknown as Dicer. siRNA duplex products are recruited into a multi-proteinsiRNA complex termed RISC (RNA Induced Silencing Complex). Withoutwishing to be bound by any particular theory, a RISC is then believed tobe guided to a target nucleic acid (suitably mRNA), where the siRNAduplex interacts in a sequence-specific way to mediate cleavage in acatalytic fashion. Small interfering RNAs that can be used accordancewith the present invention can be synthesized and used according toprocedures that are well known in the art and that will be familiar tothe ordinarily skilled artisan. Small interfering RNAs for use in themethods of the present invention suitably comprise between about 1 toabout 50 nucleotides (nt). In examples of non limiting embodiments,siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt,about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25nucleotides.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced,Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarily to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

By “enzymatic RNA” is meant an RNA molecule with enzymatic activity(Cech, (1988) J. American. Med. Assoc. 260, 3030-3035). Enzymaticnucleic acids (ribozymes) act by first binding to a target RNA. Suchbinding occurs through the target binding portion of an enzymaticnucleic acid which is held in close proximity to an enzymatic portion ofthe molecule that acts to cleave the target RNA. Thus, the enzymaticnucleic acid first recognizes and then binds a target RNA through basepairing, and once bound to the correct site, acts enzymatically to cutthe target RNA.

By “decoy RNA” is meant an RNA molecule that mimics the natural bindingdomain for a ligand. The decoy RNA therefore competes with naturalbinding target for the binding of a specific ligand. For example, it hasbeen shown that over-expression of HIV trans-activation response (TAR)RNA can act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA. Thisis meant to be a specific example. Those in the art will recognize thatthis is but one example, and other embodiments can be readily generatedusing techniques generally known in the art.

As used herein, the term “monomers” typically indicates monomers linkedby phosphodiester bonds or analogs thereof to form oligonucleotidesmailing in size from a few monomeric units, e.g., from about 3-4, toabout several hundreds of monomeric units. Analogs of phosphodiesterlinkages include: phosphorothioate, phosphorodithioate,methylphosphomates, phosphoroselenoate, phosphoramidate, and the like,as more fully described below.

The term “nucleotide” covers naturally occurring nucleotides as well asnonnaturally occurring nucleotides. It should be clear to the personskilled in the art that various nucleotides which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleotides” includes not only the known purine andpyrimidine heterocycles-containing molecules, but also heterocyclicanalogues and tautomers thereof. Illustrative examples of other types ofnucleotides are molecules containing adenine, guanine, thymine,cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in Benner et. al. U.S. Pat. No. 5,432,272. The term“nucleotide” is intended to cover every and all of these examples aswell as analogues and tautomers thereof. Especially interestingnucleotides are those containing adenine, guanine, thymine, cytosine,and uracil, which are considered as the naturally occurring nucleotidesin relation to therapeutic and diagnostic application in humans.Nucleotides include the natural 2′-deoxy and 2′-hydroxyl sugars, e.g.,as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman,San Francisco, 1992) as well as click analogs.

“Analogs” in reference to nucleotides includes synthetic nucleotideshaving modified base moieties and/or modified sugar moieties (see e.g.,described generally by Scheit, Nucleotide Analogs, John Wiley, New York1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443,Toulmé, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M.,(1999) Biochemica et Biophysica Acta 1489: 117-139; Freier S. M., (1997)Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery &Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic AcidDrug Dev., 10:297-310); 2′-O, 3′-C-linked[3.2.0]bicycloarabinonucleosides. Such analogs include syntheticnucleotides designed to enhance binding properties, e.g., duplex ortriplex stability, specificity, or the like.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of oligomeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoögsteen orreversed Hoögsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleotides) of the strands of oligomeric compounds.For example, adenine and thymine are complementary nucleotides whichpair through the formation of hydrogen bonds. Hybridization can occurunder varying circumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated. In general, stringent hybridization conditionscomprise low concentrations (<0.15M) of salts with inorganic canons suchas Na++ or K++ (i.e., low ionic strength), temperature higher than 20°C.-25° C. below the Tm of the oligomeric compound:target sequencecomplex, and the presence of denaturants such as formamide,dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecylsulfate (SDS). For example, the hybridization rate decreases 1.1% foreach 1% formamide. An example of a high stringency hybridizationcondition is 0.1× sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v)SDS at 60° C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two oligomeric strands. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, said target nucleic acid being a DNA, RNA, oroligonucleotide molecule, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the further DNA,RNA, or oligonucleotide molecule are complementary to each other when asufficient number of complementary positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedindicate a sufficient degree of precise pairing or complementarity overa sufficient number of nucleotides such that stable and specific bindingoccurs between the oligomeric compound and a target nucleic acid.

It is understood in the art that the sequence of an oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure). The oligomeric compounds of the presentinvention comprise at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which they aretargeted. For example, an antisense compound in which 18 of 20nucleotides of the antisense compound are complementary to a targetlegion, and would therefore specifically hybridize, would represent 90percent complementarily. In this example, the remaining noncomplementarynucleotides may be clustered or interspersed with complementarynucleotides and need not be contiguous to each other or to complementarynucleotides. As such, an antisense compound which is 18 nucleotides inlength having 4 (four) noncomplementary nucleotides which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art.Percent homology, sequence identity or complementarity, can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).

As used herein, the term “Thermal Melting Point (Tm)” refers to thetemperature, under defined ionic strength, pH, and nucleic acidconcentration, at which 50% of the oligonucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0M Na ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

As used herein, “modulation” means either an increase (stimulation) or adecrease (inhibition) the expression of a gene.

The term “variant”, when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation inthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ornucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs,) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, aryl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which are known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

As used herein, the term “animal” or “patient” is meant to include, forexample, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys,horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken,reptiles, fish, insects and arachnids.

“Mammal” covers warm blooded mammals that are typically under medicalcare (e.g., humans and domesticated animals). Examples include feline,canine, equine, bovine, and human, as well as just human.

“Treating” or “treatment” covers the treatment of a disease-state in amammal, and includes: (a) preventing the disease-state occurring in amammal, in particular, when such mammal is predisposed to thedisease-state but has not yet been diagnosed as having it; (b)inhibiting the disease-state, e.g., arresting it development; and/or (c)relieving the disease-state, e.g., causing regression of the diseasestate until a desired endpoint is reached. Treating also includes theamelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration may or may not be directlyaffecting the disease (e.g., cause, transmission, expression, etc.).

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant rumors found in mammals, including, but not limited to:leukemias, lymphomas, melanomas, carcinomas and sarcomas. The cancermanifests itself as a “tumor” or tissue comprising malignant cells ofthe cancer. Examples of tumors include sarcomas and carcinomas such as,but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor,lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Additional cancers which can be treated by the disclosedcomposition according to the invention include but not limited to, forexample, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myelomaneuroblastoma, breast cancer, ovarian cancer, lung cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,small-cell lung tumors, primary brain tumors, stomach cancer, coloncancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, gastric cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, cervical cancer,endometrial cancer, adrenal conical cancer, and prostate cancer.

Polynucleotide and Oligonucleotide Compositions and Molecules

Targets: In one embodiment, the targets comprise nucleic acid sequencesof Tumor Protein 63 (p63), including without limitation sense and/orantisense noncoding and/or coding sequences associated with p63.

Tumor protein 63 is a nuclear protein located on chromosome 3q, has beenshown to possess homology with p53, a tumor-suppressor gene encoding amulti-functional DNA-binding protein important in cell cycle and celldeath regulation. p63 exists in three isoforms, each of which can encodetwo categories of transcripts under the control of two alternativepromoters. The first encodes full length proteins with an N-terminaltransactivation domain that, like p53, can activate transcription andinduce apoptosis. The second encodes truncated proteins lacking theN-terminal transactivation domain (ANp63), and potentially acting in adominant-negative manner to suppress transactivation by p53 and/or fulllength p63.

In an embodiment, antisense oligonucleotides are used to prevent ortreat diseases or disorders associated with p63 family members.Exemplary Tumor Protein 63 (p63) mediated diseases and disorders whichcan be treated with cell/tissues regenerated from stem cells obtainedusing the antisense compounds comprise: a disease or disorder associatedwith abnormal function and/or expression of p63, cancer, tumor, aproliferative disease of disorder, Corneal dystrophy, prematuremenopause, a disease or disorder associated with impaired hair growthcycle (e.g., alopecia etc.), a disease or disorder associated withimpaired development of skin appendage (such as teeth, hair follicles,mammary, lacrimal and salivary glands, exocrine glands etc.),ectrodactyly-ectodermal dysplasia-cleft syndrome, Hay-Wells syndrome,limb mammary syndrome, a pulmonary disease or disorder,aero-dermato-ungual-lacrimal-tooth syndrome, nonsyndromicsplit-hand/foot malformation, isolated cleft lip/palate, Rapp-Hodgkinsyndrome, a skin disease or disorder (e.g., wound, alopecia (baldness),and a disease and disorder associated with aging and senescence.

In embodiments of the present invention, therapeutic and/or cosmeticregimes and related tailored treatments are provided to subjectsrequiring skin treatments or at risk of developing conditions for whichthey would require skin treatments. Diagnosis can be made, e.g., basedon the subject's Tumor Protein 63 status. A patient's Tumor Protein 63expression levels in a given tissue such as skin can be determined bymethods known to those of skill in the art and described elsewhereherein, e.g., by analyzing tissue using PCR antibody-based detectionmethods.

A preferred embodiment of the present invention provides a compositionfor skin treatment and/or a cosmetic application comprising TumorProtein 63 antisense oligonucleotides, e.g., to upregulate expression ofTumor Protein 63 in the skin. Examples of antisense oligonucleotides areset forth as SEQ ID NOS: 3 to 6. U.S. Pat. No. 7,544,497, “Compositionsfor manipulating the lifespan and stress response of cells andorganisms,” incorporated herein by reference, describes potentialcosmetic use for agents that modulate Tumor Protein 63 activity byreducing the K_(m) of the Tumor Protein 63 protein for its substrate. Inembodiments, cells are treated in vivo with the oligonucleotides of thepresent invention, to increase cell lifespan or prevent apoptosis. Forexample, skin can be protected from aging, e.g., developing wrinkles, bytreating skin, e.g., epithelial cells, as described herein. In anexemplary embodiment, skin is contacted with a pharmaceutical orcosmetic composition comprising a Tumor Protein 63 antisense compound asdescribed herein. Exemplary skin afflictions or skin conditions includedisorders or diseases associated with or caused by inflammation, sundamage or natural aging. For example, the compositions find utility inthe prevention or treatment of contact dermatitis (including irritantcontact dermatitis and allergic contact dermatitis), atopic dermatitis(also known as allergic eczema), actinic keratosis, keratinizationdisorders (including eczema), epidermolysis bullosa diseases (includingpenfigus), exfoliative dermatitis, seborrheic dermatitis, erythemas(including erythema multiforme and erythema nodosum), damage caused bythe sun or other light sources, discoid lupus erythematosus,dermatomyositis, skin cancer and the effects of natural aging.

In embodiments of the present invention, a composition comprising TumorProtein 63 antisense oligonucleotides, e.g., to upregulate expression ofTumor Protein 63 in the scalp and inhibit androgen receptor signaling,thereby preventing androgenetic alopecia (hair loss). In embodiments, apatient suffering from alopecia is administered either a topical orsystemic formulation.

In an embodiment, an antisense oligonucleotide described herein isincorporated into a topical formulation containing a topical carrierthat is generally suited to topical drug administration and comprisingany such material known in the art. The topical carrier may be selectedso as to provide the composition in the desired form, e.g., as anointment, lotion, cream, microemulsion, gel, oil, solution, or the like,and may be comprised of a material of either naturally occurring orsynthetic orient. It is preferable that the selected carrier notadversely affect the active agent or other components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like. Formulations may be colorless,odorless ointments, lotions, creams, microemulsions and gels.

Antisense oligonucleotides of the invention may be incorporated intoointments, which generally are semisolid preparations which aretypically based on petrolatum or other petroleum derivatives. Thespecific ointment base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery,and, preferably, will provide for other desired characteristics as well,e.g., emolliency or the like. As with other carriers or vehicles, anointment base should be inert, stable, nonirritating and nonsensitizing.As explained in Remington's Pharmaceutical Sciences (Mack Pub. Co.),ointment bases may be grouped into four classes: oleaginous bases;emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginousointment bases include, for example, vegetable oils, fats obtained fromanimals, and semisolid hydrocarbons obtained from petroleum.Emulsifiable ointment bases, also known as absorbent ointment bases,contain little or no water and include, for example, hydroxystearinsulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointmentbases are either water-in-oil (W/O) emulsions or oil-in-water (O/W)emulsions, and include, for example, cetyl alcohol, glycerylmonostearate, lanolin and stearic acid. Exemplary water-soluble ointmentbases are prepared from polyethylene glycols (PEGs) of varying molecularweight (see, e.g., Remington's, supra).

Antisense oligonucleotides of the invention may be incorporated intolotions, which generally are preparations to be applied to the skinsurface without friction, and are typically liquid or semiliquidpreparations in which solid particles, including the active agent, arepresent in a water or alcohol base. Lotions are usually suspensions ofsolids, and may comprise a liquid oily emulsion of the oil-in-watertype. Lotions are preferred formulations for treating large body areas,because of the ease of applying a more fluid composition. It isgenerally necessary that the insoluble matter in a lotion be finelydivided. Lotions will typically contain suspending agents to producebetter dispersions as well as compounds useful for localizing andholding the active agent in contact with the skin, e.g.,methylcellulose, sodium carboxymethylcellulose, or the like. Anexemplary lotion formulation for use in conjunction with the presentmethod contains propylene glycol mixed with a hydrophilic petrolatumsuch as that which may be obtained under the trademark Aquaphor.sup®from Beiersdorf, Inc, (Norwalk, Conn.).

Antisense oligonucleotides of the invention may be incorporated intocreams, which generally are viscous liquid or semisolid emulsions,either oil-in-water or water-in-oil. Cream bases are water-washable, andcontain an oil phase, an emulsifier and an aqueous phase. The oil phaseis generally comprised of petrolatum and a fatty alcohol such as cetylstearyl alcohol; the aqueous phase usually, although not necessarily,exceeds the oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation, as explained in Remington's, supra,is generally a nonionic, anionic, cationic or amphoteric surfactant.

Antisense oligonucleotides of the invention may be incorporated intomicroemulsions, which generally are thermodynamically stable,isotropically clear dispersions of two immiscible liquids, such as oiland water, stabilized by an interfacial film of surfactant molecules(Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker,1992), volume 9). For the preparation of microemulsions, surfactant(emulsifier), co-surfactant (co-emulsifier), an oil phase and a waterphase are necessary. Suitable surfactants include any surfactants thatare useful in the preparation of emulsions, e.g., emulsifiers that aretypically used in the preparation of creams. The co-surfactant (or“co-emulsifer”) is generally selected from the group of polyglycerolderivatives, glycerol derivatives and fatty alcohols. Preferredemulsifier/co-emulsifier combinations are generally although notnecessarily selected from the group consisting of: glyceryl monostearateand polyoxyethylene stearate; polyethylene glycol and ethylene glycolpalmitostearate; and caprilic and capric triglycerides and oleoylmacrogolglycerides. The water phase includes not only water but also,typically, buffers, glucose, propylene glycol, polyethylene glycols,preferably lower molecular weight polyethylene glycols (e.g., PEG 300and PEG 400), and/or glycerol, and the like, while the oil phase willgenerally comprise, for example, fatty acid esters, modified vegetableoils, silicone oils, mixtures of mono- di- and triglycerides, mono- anddi-esters of PEG (e.g., oleoyl macrogol glycerides), etc.

Antisense oligonucleotides of the invention may be incorporated into gelformulations, which generally are semisolid systems consisting of eithersuspensions made up of small inorganic particles (two-phase systems) orlarge organic molecules distributed substantially uniformly throughout acarrier liquid (single phase gels). Single phase gels can be made, forexample, by combining the active agent, a carrier liquid and a suitablegelling agent such as tragacanth (at 2 to 5%), sodium alginate (at2-10%), gelatin (at 2-15%), methylcellulose (at 3-5%), sodiumcarboxymethylcellulose (at 2-5%), carbomer (at 0.3-5%) or polyvinylalcohol (at 10-20%) together and mixing until a characteristic semisolidproduct is produced. Other suitable gelling agents includemethylhydroxycellulose, polyoxyethylene-polyoxypropylene,hydroxyethylcellulose and gelatin. Although gels commonly employ aqueouscarrier liquid, alcohols and oils can be used as the carrier liquid aswell.

Various additives, known to those skilled in the art, may be included informulations, e.g., topical formulations. Examples of additives include,but are not limited to, solubilizers, skin permeation enhancers,opacifiers, preservatives (e.g., anti-oxidants), gelling agents,buffeting agents, surfactants (particularly nonionic and amphotericsurfactants), emulsifiers, emollients, thickening agents, stabilizers,humectants, colorants, fragrance, and the like. Inclusion ofsolubilizers and/or skin permeation enhancers is particularly preferred,along with emulsifiers, emollients and preservatives. An optimum topicalformulation comprises approximately: 2 wt. % to 60 wt. %, preferably 2wt. % to 50 wt. %, solubilizer and/or skin permeation enhancer; 2 wt. %to 50 wt. %, preferably 2 wt. % to 20 wt. %, emulsifiers; 2 wt. % to 20wt. % emollient; and 0.01 to 0.2 wt. % preservative, with the activeagent and carrier (e.g., water) making of the remainder of theformulation.

A skin permeation enhancer serves to facilitate passage of therapeuticlevels of active agent to pass through a reasonably sized area ofunbroken skin. Suitable enhancers are well known in the art and include,for example: lower alkanols such as methanol ethanol and 2-propanol;alkyl methyl sulfoxides such as dimethylsulfoxide (DMSO),decylmethylsulfoxide (C.sub10 MSO) and tetradecylmethyl sulfboxide;pyrrolidones such as 2-pyrrolidone, N-methyl-2-pyrrolidone andN—(-hydroxyethyl)pyrrolidone; urea; N,N-diethyl-m-toluamide,C.sub2-C.sub6 alkanediols; miscellaneous solvents such as dimethylformamide (DMF), N,N-dimethylacetamide (DMA) and tetrahydrofurfurylalcohol; and the 1-substituted azacycloheptan-2-ones, particularly1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under thetrademark Azone.sup® from Whitby Research Incorporated, Richmond, Va.).

Examples or solubilizers include, but are not limited to, the following:hydrophilic ethers such as diethylene glycol monoethyl ether(ethoxydiglycol, available commercially as Transcutol.sup®) anddiethylene glycol monoethyl ether oleate (available commercially asSoficutol.sup®); polyethylene castor oil derivatives such as polyoxy 35castor oil, polyoxy 40 hydrogenated castor oil, etc.; polyethyleneglycol, particularly lower molecular weight polyethylene glycols such asPEG 300 and PEG 400, and polyethylene glycol derivatives such as PEG-8caprylic/capric glycerides (available commercially as Labrasol.sup®);alkyl methyl sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidoneand N-methyl-2-pyrrolidone; and DMA. Many solubilizers can also act asabsorption enhancers. A single solubilizer may be incorporated into theformulation, or a mixture of solubilizers may be incorporated therein.

Suitable emulsifiers and co-emulsifiers include, without limitation,those emulsifiers and co-emulsifiers described with respect tomicroemulsion formulations. Emollients include, for example, propyleneglycol, glycerol, isopropyl myristate, polypropylene glycol-2 (PPG-2)myristyl ether propionate, and the like.

Other active agents may also be included in formulations, e.g., otheranti-inflammatory agents, analgesics, antimicrobial agents, antifungalagents, antibiotics, vitamins, antioxidants, and sunblock agentscommonly found in sunscreen formulations including, but not limited to,anthranilates, benzophenones (particularly benzophenone-3), camphorderivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoylmethanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid(PABA) and derivatives thereof, and salicylates (e.g., octylsalicylate).

In an embodiment, modulation of p63 by one or more antisenseoligonucleotides is administered to a patient in need thereof, forathletic enhancement and body building.

In an embodiment, modulation of p63 by one or more antisenseoligonucleotides is administered to a patient in need thereof, toprevent or treat any disease or disorder related to p63 abnormalexpression, function, activity as compared to a normal control.

In an embodiment, the oligonucleotides are specific for polynucleotidesof p63, which includes, without limitation noncoding regions. The p63targets comprise variants of p63; mutants of p63, including SNPs;noncoding sequences of p63; alleles, fragments and the like. Preferablythe oligonucleotide is an antisense RNA molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to p63 polynucleotides alone but extends to anyof the isoforms, receptors, homologs, non-coding regions and the like ofp63.

In an embodiment, oligonucleotide targets a natural antisense sequence(natural antisense to the coding and non-coding regions) of p63 targets,including, without limitation, variants, alleles, homologs, mutants,derivatives, fragments and complementary sequences thereto. Preferablythe oligonucleotide is an antisense RNA or DNA molecule.

In an embodiment the oligomeric compounds of the present invention alsoinclude variants in which a different base is present at one or more ofthy nucleotide positions in the compound. For example, if the firstnucleotide is an adenine, variants may be produced which containthymidine, guanosine, cytidine or other natural or unnatural nucleotidesat this position. This may be done at any of the positions of theantisense compound. These compounds are then tested using the methodsdescribed herein to determine their ability to inhibit expression of atarget nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 50% to about60%. In some embodiments, homology, sequence identity orcomplementarity, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarity, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired. Such conditionsinclude, i.e., physiological conditions in the case of in vivo assays ortherapeutic treatment, and conditions in which assays are performed inthe case of in vitro assays.

An antisense compound, whether DNA, RNA, chimeric, substituted etc, isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarily to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

In an embodiment, targeting of p63 including without limitation,antisense sequences which are identified and expanded, using forexample, PCR, hybridization etc., one or more of the sequences set forthas SEQ ID NOS: 2, and the like, modulate the expression or function ofp363. In one embodiment, expression or function is up-regulated ascompared to a control. In an embodiment, expression or function isdown-regulated as compared to a control.

In an embodiment, oligonucleotides comprise nucleic acid sequences setfirth as SEQ ID NOS: 3 to 6 including antisense sequences which areidentified and expanded, using for example, PCR, hybridization etc.These oligonucleotides can comprise one or more modified nucleotides,shorter or longer fragments, modified bonds and the like. Examples ofmodified bonds or internucleotide linkages comprise phosphorothioatephosphorodithioate or the like. In an embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides per se, is also known and neednot be described here.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In embodiments of the present invention oligomeric antisense compounds,particularly oligonucleotides, bind to target nucleic acid molecules andmodulate the expression and/or function of molecules encoded by a targetgene. The functions of DNA to be interfered comprise, for example,replication and transcription. The functions of RNA to be interferedcomprise all vital functions such as, for example, translocation of theRNA to the site of protein translation, translation of protein from theRNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The functions may be up-regulated or inhibited depending on thefunctions desired.

The antisense compounds, include, antisense oligomeric compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, primers, probes, and otheroligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds.

Targeting an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes Tumor Protein 63 (p63).

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

In an embodiment, the antisense oligonucleotides bind to the naturalantisense sequences of Tumor Protein 63 (p63) and modulate theexpression and/or function of p63 (SEQ ID NO: 1). Examples of antisensesequences include SEQ ID NOS: 2 to 6.

In an embodiment, the antisense oligonucleotides bind to one or moresegments of Tumor Protein 63 (p63) polynucleotides and modulate theexpression and/or function of p63. The segments comprise at least fiveconsecutive nucleotides of the p63 sense or antisense polynucleotides.

In an embodiment, the antisense oligonucleotides are specific fornatural antisense sequences of p63 wherein binding of theoligonucleotides to the natural antisense sequences of p63 modulateexpression and/or function of p63.

In an embodiment, oligonucleotide compounds comprise sequences set forthas SEQ ID NOS: 3 to 6, antisense sequences which are identified andexpanded, using for example, PCR, hybridization etc Theseoligonucleotides can comprise one or more modified nucleotides, shorteror longer fragments, modified bonds and the like. Examples of modifiedbonds or internucleotide linkages comprise phosphorothioate,phosphorodithioate or the like. In an embodiment, the nucleotidescomprise a phosphorus derivative. The phosphorus derivative (or modifiedphosphate group) which may be attached to the sugar or sugar analogmoiety in the modified oligonucleotides of the present invention may bea monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theabove-noted phosphate analogs, and their incorporation into nucleotides,modified nucleotides and oligonucleotides, per se, is also known andneed not be described here.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes has a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding Tumor Protein 63 (p63), regardless of the sequence(s) of suchcodons. A translation termination codon (or “stop codon”) of a gene mayhave one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, atargeted region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Another target region includes the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene). Still another target regionincludes the 3′ untranslated region (3′UTR), known in the art to referto the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. Another target region for thisinvention is the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. In one embodiment, targeting splicesites, i.e., intron-exon junctions or exon-intron junctions, isparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. An aberrant fusion junction due to rearrangementor deletion is another embodiment of a target site. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. Introns can beeffectively targeted using antisense compounds targeted to, for example,DNA of pre-mRNA.

In an embodiment, the antisense oligonucleotides bind to coding and/ornon-coding regions of a target polynucleotide and modulate theexpression and/or function of the target molecule.

In an embodiment, the antisense oligonucleotides bind to naturalantisense polynucleotides and modulate the expression and/or function ofthe target molecule.

In an embodiment, the antisense oligonucleotides bind to sensepolynucleotides and modulate the expression and/or function of thetarget molecule.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame germane DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more than onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also embodiments of target nucleic acids.

The locations on the target nucleic acid to which the antisensecompounds hybridize are defined as at least a 5-nucleotide long portionof a target region to which an active antisense compound is targeted.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure.

Target segments 5-100 nucleotides in length comprising a stretch of atleast five (5) consecutive nucleotides selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 5 consecutive nucleotides from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleotides beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 5 to about 100 nucleotides). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 5 consecutive nucleotides from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleotides being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 5 to about 100nucleotides). One having skill in the art armed with the target segmentsillustrated herein will be able, without undue experimentation, toidentify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In embodiments of the invention the oligonucleotides bind to anantisense strand of a particular target. The oligonucleotides are atleast 5 nucleotides in length and can be synthesized so eacholigonucleotide targets overlapping sequences such that oligonucleotidesare synthesized to cover the entire length of the target polynucleotide.The targets also include coding as well as non coding regions.

In one embodiment, it is preferred to target specific nucleic acids byantisense oligonucleotides. Targeting an antisense compound to aparticular nucleic acid, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a non coding polynucleotidesuch as for example, non coding RNA (RNA).

RNAs can be classified into (1) messenger RNAs (mRNAs), which aretranslated into proteins, and (2) non-protein-coding RNAs (ncRNAs),ncRNAs comprise microRNAs, antisense transcripts and otherTranscriptional Units (TU) containing a high density of stop codons andlacking any extensive “Open Reading Frame”. Many ncRNAs appear to startfrom initiation sites in 3′ untranslated regions (3′UTRs) ofprotein-coding loci. ncRNAs are often rare and at least half of thencRNAs that have been sequenced by the FANTOM consortium seem not to bepolyadenylated. Most researchers have for obvious reasons focused onpolyadenylated mRNAs that are processed and exported to the cytoplasm.Recently, it was shown that the set of non-polyadenylated nuclear RNAsmay be very large, and that many such transcripts arise from so-calledintergenic regions. The mechanism by which ncRNAs may regulate geneexpression is by base pairing with target transcripts. The RNAs thatfunction by base pairing can be grouped into (1) cis encoded RNAs thatare encoded at the same genetic location, but on the opposite strand tothe RNAs they act upon and therefore display perfect complementarily totheir target, and (2) trans-encoded RNAs that are encoded at achromosomal location distinct from the RNAs they act upon and generallydo not exhibit perfect base-pairing potential with their targets.

Without wishing to be bound by theory, perturbation of an antisensepolynucleotide by the antisense oligonucleotides described herein canalter the expression of the corresponding sense messenger RNAs. However,this regulation can either be discordant (antisense knockdown results inmessenger RNA elevation) or concordant (antisense knockdown results inconcomitant messenger RNA reduction). In these cases, antisenseoligonucleotides can be targeted to overlapping or non-overlapping partsof the antisense transcript resulting in its knockdown or sequestration.Coding as well as non-coding antisense can be targeted in an identicalmanner and that either category is capable of regulating thecorresponding sense transcripts—either in a concordant or disconcordantmanner. The strategies that are employed in identifying newoligonucleotides for use against a target can be based on the knockdownof antisense RNA transcripts by antisense oligonucleotides or any othermeans of modulating the desired target.

Strategy 1: In the case of discordant regulation, knocking down theantisense transcript elevates the expression of the conventional (sense)gene. Should that latter gene encode for a known or putative drugtarget, then knockdown of its antisense counterpart could conceivablymimic the action of a receptor agonist or an enzyme stimulant.

Strategy 2: In the case of concordant regulation, one couldconcomitantly knock down both antisense and sense transcripts andthereby achieve synergistic reduction of the conventional (sense) geneexpression. If, for example, an antisense oligonucleotide is used toachieve knockdown, then this strategy can be used to apply one antisenseoligonucleotide targeted to the sense transcript and another antisenseoligonucleotide to the corresponding antisense transcript, or aenergetically symmetric antisense oligonucleotide that simultaneouslytargets overlapping sense and antisense transcripts.

According to the present invention, antisense compounds includeantisense oligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, and otheroligomeric compounds which hybridize to at least a portion or the targetnucleic acid and modulate its function. As such, they may be DNA, RNA,DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one ormore of these. These compounds may be single-stranded, doublestranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges, mismatches or loops.Antisense compounds are routinely prepared linearly but can be joined orotherwise prepared to be circular and/or branched. Antisense compoundscan include constructs such as, for example, two strands hybridized toform a wholly or partially double-stranded compound or a single strandwith sufficient self-complementarity to allow for hybridization andformation of a fully or partially double-stranded compound. The twostrands can be linked internally leaving free 3′ or 5′ termini or can belinked to form a continuous hairpin structure or loop. The hairpinstructure may contain an overhang on either the 5′ or 3′ terminusproducing an extension of single stranded character. The double strandedcompounds optionally can include overhangs on the ends. Furthermodifications can include conjugate groups attached to one of thetermini, selected nucleotide positions, sugar positions or to one of theinternucleoside linkages. Alternatively, the two strands can be linkedvia a non-nucleic acid moiety or linker group. When formed front onlyone strand, dsRNA can take the form of a self-complementary hairpin-typemolecule that doubles back on itself to form a duplex. Thus, the dsRNAscan be fully or partially double stranded. Specific modulation of geneexpression can be achieved by stable expression of dsRNA hairpins intransgenic cell lines, however, in some embodiments, the gene expressionor function is up regulated. When formed from two strands, or a singlestrand that takes the form of a self-complementary hairpin-type moleculedoubled back on itself to form a duplex, the two strands (orduplex-forming regions of a single strand) are complementary RNA strandsthat base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy stunt's and, generally, T rather than U bases) or“RNA-like” (i.e., generally baying one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain both A- and B-form regions.

In an embodiment, the desired oligonucleotides or antisense compounds,comprise at least one of: antisense RNA, antisense DNA, chimericantisense oligonucleotides, antisense oligonucleotides comprisingmodified linkages, interference RNA (RNAi), short interfering RNA(siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced geneactivation (RNAa); small activating RNAs (saRNAs), or combinationsthereof.

dsRNA can also activate gene expression, a mechanism that has beentermed “small RNA-induced gene activation” or RNAa. dsRNAs targetinggene promoters induce potent transcriptional activation of associatedgenes. RNAa was demonstrated in human cells using synthetic dsRNAs,termed “small activating RNAs” (saRNAs). It is currently not knownwhether RNAa is conserved in other organisms.

Small double-stranded RNA (dsRNA), such as small interfering RNA (siRNA)and microRNA (miRNA), have been found to be the trigger of anevolutionary conserved mechanism known as RNA interference (RNAi). RNAiinvariably leads to gene silencing via remodeling chromatin to therebysuppress transcription, degrading complementary mRNA, or blockingprotein translation. However, in instances described in detail in theexamples section which follows, oligonucleotides are shown to increasethe expression and/or function of the tumor Protein 63 (p63)polynucleotides and encoded products thereof. dsRNAs may also act assmall activating RNAs (saRNA). Without wishing to be bound by theory, bytargeting sequences in gene promoters, saRNAs would induce target geneexpression in a phenomenon referred to dsRNA-induced transcriptionalactivation (RNAa).

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of Tumor Protein 63 (p63) polynucleotides.“Modulators” are those compounds that decrease or increase theexpression of a nucleic acid molecule encoding p63 and which comprise atleast a 5-nucleotide portion that is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encoding sense ornatural antisense polynucleotides of p63 with one or more candidatemodulators, and selecting for one or more candidate modulators whichdecrease or increase the expression of a nucleic acid molecule encodingp63 polynucleotides, e.g. SEQ ID NOS: 3 to 6. Once it is shown that thecandidate modulator or modulators are capable of modulating (e.g. eitherdecreasing or increasing) the expression of a nucleic acid moleculeencoding p63 polynucleotides, the modulator may then be employed infurther investigative studies of the function of p63 polynucleotides, orfor use as a research, diagnostic, or therapeutic agent in accordancewith the present invention.

Targeting the natural antisense sequence preferably modulates thefunction of the target gene. For example, the p63 gene (e.g. accessionnumber NM_001114979). In an embodiment, the target is an antisensepolynucleotide of the p63 gene. In an embodiment, an antisenseoligonucleotide targets sense and/or natural antisense sequences of p63polynucleotides (e.g. accession number NM_001114979), variants, alleles,isoforms, homologs, mutants, derivatives, fragments and complementarysequences thereto. Preferably the oligonucleotide is an antisensemolecule and the targets include coding and noncoding regions ofantisense and/or sense p63 polynucleotides.

The preferred tarter segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications. For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target.

In an embodiment, an antisense oligonucleotide targets Tumor Protein 63(p63) polynucleotides (e.g. accession number NM_001114979), variants,alleles, isoforms, homologs, mutants, derivatives, fragments andcomplementary sequences thereto. Preferably the oligonucleotide is anantisense molecule.

In accordance with embodiments of the invention, the target nucleic acidmolecule is not limited to p63 alone but extends to any of the isoforms,receptors, homologs and the like of p63 molecules.

In an embodiment, an oligonucleotide targets a natural antisensesequence of p63 polynucleotides, for example, polynucleotides set forthas SEQ ID NOS: 2, and any variants, alleles, homologs, mutants,derivatives, fragments and complementary sequences thereto. Examples ofantisense oligonucleotides are set forth as SEQ ID NOS: 3 to 6.

In one embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of p63 antisense, including without limitationnoncoding sense and/or antisense sequences associated with p63polynucleotides and modulate expression and/or function of p63molecules.

In an embodiment, the oligonucleotides are complementary to or bind tonucleic acid sequences of p63 natural antisense, set forth as SEQ IDNOS: 2 and modulate expression and/or function of p63 molecules.

In an embodiment, oligonucleotides comprise sequences of at least 5consecutive nucleotides of SEQ ID NOS: 3 to 6 and modulate expressionand/or function of p63 molecules.

The polynucleotide targets comprise p63, including family membersthereof, variants of p63; mutants of p63, including SNPs; noncodingsequences of p63 alleles of p63; species variants, fragments and thelike. Preferably the oligonucleotide is an antisense molecule.

In an embodiment, the oligonucleotide targeting p63 polynucleotides,comprise: antisense RNA, interference RNA (RNAi), short interfering RNA(siRNA); micro interfering RNA (miRNA); a small, temporal RNA (stRNA);or a short, hairpin RNA (shRNA); small RNA-induced gene activation(RNAa); or, small activating RNA (saRNA).

In an embodiment, targeting of Tumor Protein 63 (p63) polynucleotides,e.g. SEQ ID NOS: 2 modulate the expression or function of these targets.In one embodiment, expression or function is up-regulated as compared toa control. In an embodiment, expression or function is down-regulated ascompared to a control.

In an embodiment, antisense compounds comprise sequences set forth asSEQ ID NOS: 3 to 6. The oligonucleotides can comprise one or moremodified nucleotides, shorter or longer fragments, modified bonds andthe like.

In an embodiment, SEQ ID NOS: 3 to 6 comprise one or more LNAnucleotides.

The modulation of a desired target nucleic acid can be carried out inseveral ways known in the art. For example, antisense oligonucleotides,siRNA etc. Enzymatic nucleic acid molecules (e.g., ribozymes) arenucleic acid molecules capable of catalyzing one or more of a variety ofreactions, including the ability to repeatedly cleave other separatenucleic acid molecules in a nucleotide base sequence-specific manner.Such enzymatic nucleic acid molecules can be used, for example, totarget virtually any RNA transcript.

Because of their sequence-specificity, trans-cleaving enzymatic nucleicacid molecules show promise as therapeutic agents for human disease(Usman & McSwiggen, (1995) Ann. Rep. Med. Chem. 30, 285-294;Christoffersen and Marr, (1995) J. Med. Chem. 38, 2023-2037). Enzymaticnucleic acid molecules can be designed to cleave specific RNA targetswithin the background of cellular RNA. Such a cleavage event renders themRNA non-functional and abrogates protein expression from that RNA. Inthis manner, synthesis of a protein associated with a disease state canbe selectively inhibited.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closoproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, (1979) Proc. R. Soc. London, B 205, 435) have been used toevolve new nucleic acid catalysts capable of catalyzing a variety ofreactions, such as cleavage and ligation of phosphodiester linkages andamide linkages.

The development of ribozymes that are optimal for catalytic activitywould contribute significantly to any strategy that employs RNA-cleavingribozymes for the purpose of regulating gene expression. The hammerheadribozyme, for example, functions with a catalytic rate (kcat) of about 1min-1 in the presence of saturating (10 mM) concentrations of Mg2+cofactor. An artificial “RNA ligase” ribozyme has been shown to catalyzethe corresponding self-modification reaction with a rate of about 100min-1. In addition, it is known that certain modified hammerheadribozymes that have substrate binding arms made of DNA catalyze RNAcleavage with multiple turn-over rates that approach 100 min-1. Finally,replacement of a specific residue within the catalytic core of thehammerhead with certain nucleotide analogues gives modified ribozymesthat show as much as a 10-fold improvement in catalytic rate. Thesefindings demonstrate that ribozymes can promote chemical transformationswith catalytic rates that are significantly greater than those displayedin vitro by most natural self-cleaving ribozymes. It is then possiblethat the structures of certain selfcleaving ribozymes may be optimizedto give maximal catalytic activity, or that entirely new RNA motifs canbe made that display significantly faster rates for RNA phosphodiestercleavage.

Intermolecular cleavage of an RNA substrate by an RNA catalyst that fitsthe “hammerhead” model was first shown in 1987 (Uhlenbeck, O. C. (1987)Nature, 328: 596-600). The RNA catalyst was recovered and reacted withmultiple RNA molecules, demonstrating that it was truly catalytic.

Catalytic RNAs designed based on the “hammerhead” motif have been usedto cleave specific target sequences by making appropriate base changesin the catalytic RNA to maintain necessary base pairing with the targetsequences. This has allowed use of the catalytic RNA to cleave specifictarget sequences and indicates that catalytic RNAs designed according tothe “hammerhead” model may possibly cleave specific substrate RNAs invivo.

RNA interference (RNAi) has become a powerful tool for modulating geneexpression in mammals and mammalian cells. This approach requires thedelivery of small interfering RNA (siRNA) either as RNA itself or asDNA, using an expression plasmid or virus and the coding sequence forsmall hairpin RNAs that are processed to siRNAs. This system enablesefficient transport of the pre-siRNAs to the cytoplasm where they areactive and permit the use of regulated and tissue specific promoters forgene expression.

In an embodiment, an oligonucleotide or antisense compound comprises anoligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleicacid (DNA), or a mimetic, chimera, analog or homolog thereof. This termincludes oligonucleotides composed of naturally occurring nucleotides,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftendesired over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

According to the present invention, the oligonucleotides or “antisensecompounds” include antisense oligonucleotides (e.g. RNA, DNA, mimetic,chimera, analog or homolog thereof), ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, andother oligomeric compounds which hybridize to at least a portion of thetarget nucleic acid and modulate its function. As such, they may be DNA,RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of oneor more of these. These compounds may be single-stranded,double-stranded, circular or hairpin oligomeric compounds and maycontain structural elements such as internal or terminal bulges,mismatches or loops. Antisense compounds are routinely prepared linearlybut can be joined or otherwise prepared to be circular and/or branched.Antisense compounds can include constructs such as, for example, twostrands hybridized to form a wholly or partially double-strandedcompound or a single strand with sufficient self-complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded compound. The two strands can be linked internallyleaving free 3′ or 5′ termini or can be linked to form a continuoushairpin structure or loop. The hairpin structure may contain an overhangon either the 5′ or 3′ terminus producing an extension of singlestranded character. The double stranded compounds optionally can includeoverhangs on the ends. Further modifications can include conjugategroups attached to one of the termini, selected nucleotide positions,sugar positions or to one of the internucleoside linkages.Alternatively, the two strands can be linked via a non-nucleic acidmoiety or linker group. When formed from only one strand, dsRNA can takethe form of a self-complementary hairpin-type molecule that doubles backon itself to form a duplex. Thus, the dsRNAs can be fully or partiallydouble stranded. Specific modulation of gene expression can be achievedby stable expression of dsRNA hairpins in transgenic cell lines. Whenformed from two strands, or a single strand that takes the form of aself-complementary hairpin-type molecule doubled back on itself to forma duplex, the two strands (or duplex-forming regions of a single strand)are complementary RNA strands that base pair in Watson-Crick fashion.

Once introduced to a system, the compounds of the invention may elicitthe action of one or more enzymes or structural proteins to effectcleavage or other modification of the target nucleic acid or may workvia occupancy-based mechanisms. In general, nucleic acids (includingoligonucleotides) may be described as “DNA-like” (i.e., generally havingone or more 2′-deoxy sugars and, generally, T rather than U bases) or“RNA-like” (i.e., generally having one or more 2′-hydroxyl or2′-modified sugars and, generally U rather than T bases). Nucleic acidhelices can adopt more than one type of structure, most commonly the A-and B-forms. It is believed that, in general, oligonucleotides whichhave B-form-like structure are “DNA-like” and those which haveA-formlike structure are “RNA-like.” In some (chimeric) embodiments, anantisense compound may contain bath A- and B-form regions.

The antisense compounds in accordance with this invention can comprisean winsome portion from about 5 to about 80 nucleotides (i.e. from about5 to about 80 linked nucleosides) in length. This refers to the lengthof the antisense strand or portion of the antisense compound. In otherwords, a single-stranded antisense compound of the invention comprisesfrom 5 to about 80 nucleotides, and a double-stranded antisense compoundof the invention (such as a dsRNA, for example) comprises a sense and anantisense strand or portion of 5 to about 80 nucleotides in length. Oneof ordinary skill in the art will appreciate that this comprehendsantisense portions of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length, or any rangetherewithin.

In one embodiment, the antisense compounds of the invention haveantisense portions of 10 to 50 nucleotides in length. One havingordinary skill in the art will appreciate that this embodiesoligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the oligonucleotides are 15 nucleotides in length.

In one embodiment, the antisense or oligonucleotide compounds of theinvention have antisense portions of 12 or 13 to 30 nucleotides inlength. One having ordinary skill in the art will appreciate that thisembodies antisense compounds having antisense portions of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length, or any range therewithin.

In an embodiment, the oligomeric compounds of the present invention alsoinclude variants in which a different base is present at one or more ofthe nucleotide positions in the compound. For example, if the firstnucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the antisense or dsRNA compounds. Thom compoundsare then tested using the methods described herein to determine theirability to inhibit expression of a target nucleic acid.

In some embodiments, homology, sequence identity or complementarity,between the antisense compound and target is from about 40% to about60%. In some embodiments, homology, sequence identity orcomplementarily, is from about 60% to about 70%. In some embodiments,homology, sequence identity or complementarity, is from about 70% toabout 80%. In some embodiments, homology, sequence identity orcomplementarily, is from about 80% to about 90%. In some embodiments,homology, sequence identity or complementarity, is about 90%, about 92%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100%.

In an embodiment, the antisense oligonucleotides, such as for example,nucleic acid molecules set forth in SEQ ID NOS: 2 to 6 comprise one ormore substitutions or modifications. In one embodiment, the nucleotidesare substituted with locked nucleic acids (LNA).

In an embodiment, the oligonucleotides target one or more regions of thenucleic acid molecules sense and/or antisense of coding and/ornon-coding sequences associated with p63 and the sequences set forth asSEQ ID NOS: 1 and 2. The oligonucleotides are also targeted tooverlapping regions of SEQ ID NOS: 1 and 2.

Certain preferred oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the target) and a region thatis a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense modulation of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. In one an embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in thiscase, a nucleic acid encoding ms) is routinely determined by measuringthe Tm of an oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and target dissociate; dissociation isdetected spectrophotometrically. The higher the Tm, the greater is theaffinity of the oligonucleotide for the target.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotides mimetics as described above.Such; compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are nor limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In an embodiment, the region of the oligonucleotide which is modifiedcomprises at least one nucleotide modified at the 2′ position of thesugar, most preferably a 2′-Oalkyl, 2′-O-alkyl-O-alkyl or2′-fluoro-modified nucleotide. In other an embodiment, RNA modificationsinclude 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the riboseof pyrimidines, abasic residues or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than; 2′-deoxyoligonucleotides against agiven target. The effect of such increased affinity is to greatlyenhance RNAi oligonucleotide inhibition of gene expression. RNAse H is acellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes;activation of this enzyme therefore results in cleavage of the RNAtarget, and thus can greatly enhance the efficiency of RNAi inhibition.Cleavage of the RNA target can be routinely demonstrated by gelelectrophoresis. In an embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned for thisinvention include those comprising modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH, —N(CH3)-O—CH2 [known as amethylene(methylimino) or MMI backbone], CH2-O—N(CH3)-CH2,CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,). The amidebackbones disclosed by De Mesmaeker et al, (1993) Acc. Chem. Res.28:366-374 are also preferred. Also preferred are oligonucleotideshaving morpholino backbone structures (Summerton and Weller, U.S. Pat.No. 5,034,506). In other an embodiment, such as the peptide nucleic acid(PNA) backbone, the phosphodiester backbone of the oligonucleotide isreplaced with a polyamide backbone, the nucleotides being bound directlyor indirectly to the aza nitrogen atoms of the polyamide backbone.Oligonucleotides may also comprise one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3,O(CH2)nNH2 or O(CH2)nCH3 where n is from 1 to about 10; C1 to C10 loweralkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl;Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3;SO2CH3; ONO2; NO2; N3, NH2; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a reporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy [2′-O—CH2CH2OCH3, also known as2′-O-(2-methoxyethyl)]. Other preferred modifications include 2′-methoxy(2′-O—CH3), 2′-propoxy (2′-OCH2CH2CH3) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ positron of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as “base”) modificationsor substitutions. As used herein, “unmodified” or “natural” nucleotidesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleotides include nucleotides found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleotides, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and2,6-diaminopurine. A “universal” base known in the art, e.g., inosine,may be included, 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C., and are presentlypreferred base substitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety, analiphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or apolyethylene glycol chain, or Adamantane acetic acid. Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. Nos.5,138,045, 5,218,105 and 5,459,255.

It is not necessary for all positions in a given oligonucleotide to beuniformity modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

In another embodiment, the nucleic acid molecule of the presentinvention is conjugated with another moiety including but not limited toabasic nucleotides, polyether, polyamine, polyamides, peptides,carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in theart will recognize that these molecules can be linked to one or more ofany nucleotides comprising the nucleic acid molecule at severalpositions on the sugar, base or phosphate group.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of one of ordinary skill in the art. It is alsowell known to use similar techniques to prepare other oligonucleotidessuch as the phosphorothioates and alkylated derivatives. It is also wellknown to use similar techniques and commercially available modifiedamidites and controlled-pore glass (CPG) products such as biotin,fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling Va.) to synthesize fluorescentlylabeled, biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

In accordance with the invention, use of modifications such as the useof LNA monomers to enhance the potency, specificity and duration ofaction and broaden the routes of administration of oligonucleotidescomprised of current chemistries such as MOE, ANA, FANA, PS etc. Thiscan be achieved by substituting some of the monomers in the currentoligonucleotides LNA monomers. The LNA modified oligonucleotide may havea size similar to the parent compound or may be larger or preferablysmaller. It is preferred that such LNA-modified oligonucleotides containless than about 70%, more preferably less than about 60%, mostpreferably less than about 50% LNA monomers and that their sizes arebetween about 5 and 25 nucleotides, more preferably between about 12 and20 nucleotides.

Preferred modified oligonucleotide backbones comprise, but not limitedto, phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5″-2′. Varioussalts, mixed salts and free acid forms an also included.

Representative United States patents that teach the preparation of theabove phosphorus containing linkages comprise, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each or which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholine linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones, alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides comprise, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds comprise, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen, et al. (1991) Science 254, 1497-1500.

In an embodiment of the invention the oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH2-NH—O—CH2-, —CH2-N(CH3)-O—CH2- known asa methylene (methylimino) or MMI backbone, —CH2-O—N(CH3)-CH2-,—CH2N(CH3)-N(CH3)CH2- and —O—N(CH3)-CH2-CH2- wherein the nativephosphodiester backbone is represented as —O—P—O—CH2- of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—,S—or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or substituted C to CO alkyl or C2 to COalkenyl and alkynyl. Particularly preferred are O(CH2)n OmCH3, O(CH2)n,OCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2nON(CH2)nCH3)2 wheren and m can be from 1 to about 10. Other preferred oligonucleotidescomprise one of the following at the 2′ position: C to CO, (lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification comprises 2′-methoxyethoxy (2′-O—CH2CH2OCH3,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) i.e., an alkoxyalkoxygroup. A further preferred modification comprises2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as2-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2.

Other preferred modifications comprise 2′-methoxy (2′-O CH3),2′-aminopropoxy (2′-O CH2CH2CH2NH2) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures comprise, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

Oligonucleoties may also comprise nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleotides comprise the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleotides comprise other synthetic andnatural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleotides comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., ‘AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, ‘Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleotides are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Thesecomprise 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and0-6 substituted purines, comprising 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Researchand Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-Omethoxyethyl sugar modifications.

Representative United States patents that teach the preparation of theabove noted modified nucleotides as well as other modified nucleotidescomprise, but are not limited to, U.S. Pat. No. 3,687,808, as well asU.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941,each of which is herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide.

Such moieties comprise but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or Adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates comprise, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Drug Discovery: The compounds of the present invention can also beapplied in the areas of drug discovery and larger validation. Thepresent invention comprehends the use of the compounds and preferredtarget segments identified herein in drug discovery efforts to elucidaterelationships that exist between Tu or Protein 63 (p63) polynucleotidesand a disease state, phenotype, or condition. These methods includedetecting or modulating p63 polynucleotides comprising contacting asample, tissue, cell, or organism with the compounds of the presentinvention, measuring the nucleic acid or protein level of p63polynucleotides and/or a related phenotypic or chemical endpoint at sometime after treatment, and optionally comparing the measured value to anon-treated sample or sample treated with a further compound of theinvention. These methods can also be performed in parallel or incombination with other experiments to determine the function of unknowngenes for the process of target validation or to determine the validityof a particular gene product as a target for treatment or prevention ofa particular disease, condition, or phenotype.

Assessing Up-Regulation or Inhibition of Gene Expression:

Transfer of an exogenous nucleic acid into a host cell or organism canbe assessed by directly detecting the presence of the nucleic acid inthe cell or organism. Such detection can be achieved by several methodswell known in the art. For example, the presence of the exogenousnucleic acid can be detected by Southern blot or by a polymerase chainreaction (PCR) technique using primers that specifically amplifynucleotide sequences associated with the nucleic acid. Expression of theexogenous nucleic acids can also be measured using conventional methodsincluding gene expression analysis. For instance, mRNA produced from anexogenous nucleic acid can be detected and quantified using a Northernblot and reverse transcription PCR (RT-PCR).

Expression of RNA from the exogenous nucleic acid can also be detectedby measuring an enzymatic activity or a reporter protein activity. Forexample, antisense modulatory activity can be measured indirectly as adecrease or increase in target nucleic acid expression as an indicationthat the exogenous nucleic acid is producing the effector RNA. Based onsequence conservation, primers can be designed and used to amplifycoding regions of the target genes. Initially, the most highly expressedcoding region from each gene can be used to build a model control gene,although any coding or non coding region can be used. Each control geneis assembled by inserting each coding region between a reporter codingregion and its poly(A) signal. These plasmids would produce an mRNA witha reporter gene in the upsteam portion of the gene and a potential RNAitarget in the 3′ non-coding region. The effectiveness of individualantisense oligonucleotides would be assayed by modulation of thereporter gene. Reporter genes useful in the methods of the presentinvention include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracycline. Methods to determine modulation of areporter gene are well known in the art, and include, but are notlimited to, fluorometric methods (e.g. fluorescence spectroscopy,Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy),antibiotic resistance determination.

p63 protein and mRNA expression can be assayed using methods known tothose of skill in the art and described elsewhere herein. For example,immunoassays such as the ELISA can be used to measure protein levels.p63 ELISA assay kits are available commercially, e.g., from R&D Systems(Minneapolis, Minn.).

In embodiments, p63 expression (e.g., mRNA or protein) in a sample(e.g., cells or tissues in vivo or in vitro) treated using an antisenseoligonucleotide of the invention is evaluated by comparison with p63expression in a control sample. For example, expression of the proteinor nucleic acid can be compared using methods known to those of skill inthe art with that in a mock-treated or untreated sample. Alternatively,comparison with a sample treated with a control antisenseoligonucleotide (e.g., one having an altered or different sequence) canbe made depending on the information desired. In another embodiment, adifference in the expression of the p63 protein or nucleic acid in atreated vs. an untreated sample can be compared with the difference inexpression of a different nucleic acid (including any standard deemedappropriate by the researcher, e.g., a housekeeping gene) in a treatedsample vs. an untreated sample.

Observed differences can be expressed as desired, e.g., in the form of aratio or fraction, for use in a comparison with control. In embodiments,the level of p63 mRNA or protein, in a sample treated with an antisenseoligonucleotide of the present invention, is increased or decreased byabout 1.25-fold to about 10-fold or more relative to an untreated sampleor a sample treated with a control nucleic acid. In embodiments, thelevel of p63 mRNA or protein is increased or decreased by at least about1.25-fold, at least about 1.3-fold, at least about 1.4-fold, at leastabout 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, atleast about 1.8-fold, at least about 2-fold, at least about 2.5-fold, atleast about 3-fold, at least about 3.5-fold, at least about 4-fold, atleast about 4.5-fold, at least about 5-fold, at least about 5.5-fold, atleast about 6-fold, at least about 6.5-fold, at least about 7-fold, atleast about 7.5-fold, at least about 8-fold, at least about 8.5-fold, atleast about 9-fold, at least about 9.5-fold, or at least about 10-foldor more.

Kits Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, and prophylaxis, and as research reagents and componentsof kits. Furthermore, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes orto distinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics and in various biological systems, thecompounds of the present invention, either alone or in combination withother compounds or therapeutics are useful as tools in differentialand/or combinatorial analyses to elucidate expression patters of aportion or the entire complement of genes expressed within cells andtissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetent to express products of the Tumor Protein 63 (p63) genes. Theseinclude, but are not limited to, humans, transgenic animals, cells, cellcultures, tissues, xenografts, transplants and combinations thereof.

As one non limiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays, SAGE (serial analysis of gene expression),READS (restriction enzyme amplification of digested cDNAs), TOGA (totalgene expression analysis), protein arrays and proteomics, expressedsequence is (EST) sequencing, subtractive RNA fingerprinting (SuRF),subtractive cloning, differential display (DD), comparative genomichybridization, FISH (fluorescent in situ hybridization) techniques andmass spectrometry methods.

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding TumorProtein 63 (p63). For example, oligonucleotides that hybridize with suchefficiency and under such conditions as disclosed herein as to beeffective p63 modulators are effective primers or probes underconditions favoring gene amplification or detection, respectively. Theseprimes and probes are useful in methods requiring the specific detectionof nucleic acid molecules encoding p63 and in the amplification of saidnucleic acid molecules for detection or for use in further studies ofp63. Hybridization of the antisense oligonucleotides, particularly theprimers and probes, of the invention with a nucleic acid encoding p63can be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabeling of theoligonucleotide, or any other suitable detection means. Kits using suchdetection means for detecting the level of p63 in a sample may also beprepared.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs have beensafely and effectively administered to humans and numerous clinicaltrials are presently underway. It is thus established that antisensecompounds can be useful therapeutic modalities that can be configured tobe useful in treatment regimes for the treatment of cells, tissues andanimals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofp63 polynucleotides is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment a therapeutically effective amount of p63modulator. The p63 modulators of the present invention effectivelymodulate the activity of the p63 or modulate the expression of the p63protein. In one embodiment, the activity or expression of p63 in ananimal is inhibited by about 10% as compared to a control. Preferably,the activity or expression of p63 in an animal is inhibited by about30%. More preferably, the activity or expression of p63 in an animal isinhibited by 50% or more. Thus, the oligomeric compounds modulateexpression of Tumor Protein 63 (p63) mRNA by at least 10%, by at least50%, by at least 25%, by at least 30%, by a least 40%, by at least 50%,by at least 60%, by at least 70%, by at least 75%, by at least 80%, byat least 85%, by at least 90%, by at least 95%, by at least 98%, by atleast 99% or by 10% as compared to a control.

In one embodiment, the activity or expression of Tumor Protein 63 (p63)and/or in an animal is increased by about 10% as compared to a control.Preferably, the activity or expression of p63 in an animal is increasedby about 30%. More preferably, the activity or expression of p63 in ananimal is increased by 50% or more. Thus, the oligomeric compoundsmodulate expression of p63 mRNA by at least 10%, by at least 50%, by atleast 25%, by at least 30%, by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by a least 75%, by at least 80%, by at least85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%,or by 100% as compared to a control.

For example, the reduction of the expression of Tumor Protein 63 (p63)may be measured in serum, blood, adipose tissue, liver or any other bodyfluid, tissue or organ of the animal. Preferably, the cells containedwithin said fluids, tissues or organs being analyzed contain a nucleicacid molecule encoding p63 peptides and/or the p63 protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking so the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmcokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, which are incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether. e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate, a polyamine or apolyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

Representative United States patents that teach the preparation of sucholigonucleotides conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

Although, the antisense oligonucleotides do not need to be administeredin die context of a vector in order to modulate a target expressionand/or function, embodiments of the invention relates to expressionvector constructs for the expression of antisense oligonucleotides,comprising promoters, hybrid promoter gene sequences and possess astrong constitutive promoter activity, or a promoter activity which canbe induced in the desired case.

In an embodiment, invention practice involves administering at least oneof the foregoing antisense oligonucleotides with a suitable nucleic aciddelivery system. In one embodiment, that system includes a non-viralvector operably linked to the polynucleotide. Examples of such nonviralvectors include the oligonucleotide alone (e.g. any one or more of SEQID NOS: 3 to 6) or in combination with a suitable protein,polysaccharide or lipid formulation.

Additionally suitable nucleic acid delivery systems include viralvector, typically sequence from at least one of an adenovirus,adenovirus-associated virus (AAV), helper-dependent adenovirus,retrovirus, or hemagglutinatin virus of Japan-liposome (HVJ) complex.Preferably, the viral vector comprises a strong eukaryotic promoteroperably linked to de polynucleotide e.g., a cytomegalovirus (CMV)promoter.

Additionally preferred vectors include viral vectors, fusion proteinsand chemical conjugates. Retroviral vectors include Moloney murineleukemia viruses and HIV-based viruses. One preferred HIV-based viralvector comprises at least two vectors wherein the gag and pol genes arefrom an HIV genome and the env gene is from another virus. DNA viralvectors are preferred. These vectors include pox vectors such asorthopox or avipox vectors, herpesvirus vectors such as a herpes simplexI virus (HSV) vector, Adenovirus Vectors and Adeno-associated VirusVectors.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that main the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein by reference.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

For treating tissues in the central nervous system, administration canbe made by, e.g., injection or infusion into the cerebrospinal fluid.Administration of antisense RNA into cerebrospinal fluid is described,e.g., in U.S. Pat. App. Pub. No. 2007/0117772, “Methods for slowingfamilial ALS disease progression,” incorporated herein by reference inits entirety.

When it is intended that the antisense oligonucleotide of the presentinvention be administered to cells in the central nervous system,administration can be with one or more agents capable of promotingpenetration of the subject antisense oligonucleotide across theblood-brain barrier. Injection can be made, e.g., in the entorhinalcortex or hippocampus. Delivery of neurotrophic factors byadministration of an adenovirus vector to motor neurons in muscle tissueis described in, e.g., U.S. Pat. No. 6,632,427,“Adenoviral-vector-mediated gene transfer into medullary motor neurons,”incorporated herein by reference. Delivery of vectors directly to thebrain, e.g., the striatum, the thalamus, the hippocampus, or thesubstantia nigra, is known in the art and described, e.g., in U.S. Pat.No. 6,756,523, “Adenovirus vectors for the transfer of foreign genesinto cells of the central nervous system particularly in brain,”incorporated herein by reference. Administration can be rapid as byinjection or made over a period of time as by slow infusion oradministration of slow release formulations.

The subject antisense oligonucleotides can also be linked or conjugatedwith agents that provide desirable pharmaceutical or pharmacodynamicproperties. For example, the antisense oligonucleotide can be coupled toany substance, known in the art to promote penetration or transportacross the blood-brain barrier, such as an antibody to the transferrinreceptor, and administered by intravenous injection. The antisensecompound can be linked with a viral vector, for example, that makes theantisense compound more effective and/or increases the transport of theantisense compound across the blood-brain barrier. Osmotic blood brainbarrier disruption can also be accomplished by, e.g. infusion of sugarsincluding, but not limited to, meso erythritol, xylitol, D(+) galactose,D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(−) fructose, D(−)mannitol, D(+) glucose, D(+) arabinose, D(−) arabinose, cellobiose, D(+)maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(−) ribose,adonitol, D(+) arabitol, L(−) arabitol, D(+) fucose, L(−) fucose, D(−)lyxose, L(+) lyxose, and L(−) lyxose, or amino acids including, but notlimited to, glutamine, lysine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glycine, histidine, leucine, methionine,phenylalanine, proline, serine, threonine, tyrosine, valine, andtaurine. Methods and materials for enhancing blood brain barrierpenetration are described, e.g., in U.S. Pat. No. 4,866,042, “Method forthe delivery of genetic material across the blood brain barrier,” U.S.Pat. No. 6,294,520, “Material for passage through the blood-brainbarrier,” and U.S. Pat. No. 6,936,589, “Parenteral delivery systems,”all incorporated herein by reference in their entirety.

The subject antisense compounds may be admixed, encapsulated, conjugatedor otherwise associated with other molecules, molecule structures ormixtures of compounds, for example, liposomes, receptor-targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. For example, cationic lipids maybe included in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is LIPOFECTIN (availablefrom GIBCO-BRL, Bethesda, Md.).

Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogeneous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes that are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in liposomes with enhanced circulation lifetimes relative toliposomes lacking such specialized lipids. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome comprises one or more glycolipids or isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. Liposomes and their uses are furtherdescribed in U.S. Pat. No. 6,287,860.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating nonsurfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein by reference.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine,dimyristoylphosphphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andshelters. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein by reference. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhances include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein by reference.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bischloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone; tamoxifen,dacarbazine, procarbozine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drags,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. For example, the first targetmay be a particular antisense sequence of Tumor Protein 63 (p63), andthe second target may be a region from another nucleotide sequence.Alternatively, compositions of the invention may contain two or moreantisense compounds targeted to different regions of the same TumorProtein 63 (p63) nucleic acid target. Numerous examples of antisensecompounds are illustrated herein and others may be selected from amongsuitable compounds known in the art. Two or more combined compounds maybe used together or sequentially.

Dosing:

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonuclotides,and can generally be estimated based on EC50s found to be effective invitro and in vivo animal models. In general, dosage is from 0.01 μg to100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the can easily estimate repetition rates for dosingbased on measured residence times and concentrations of the drug inbodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

In embodiments, a patient is treated with a dosage of drug that is atleast about 1, at least about 2, at least about 3, at least about 4, atleast about 5, at least about 6, at least about 7, at least about 8, atleast about 9, at least about 10, at least about 15, at least about 20,at least about 25, at least about 30, at least about 35, at least about40, at least about 45, at least about 50, at least about 60, at leastabout 70, at least about 80, at least about 90, or at least about 100mg/kg body weight. Certain injected dosages of antisenseoligonucleotides are described, e.g., in U.S. Pat. No. 7,563,884,“Antisense modulation of PTPIB expression,” incorporated herein byreference in its entirety.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Numerous changes to the disclosedembodiments can be made in accordance with the disclosure herein withoutdeparting from the spirit or scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove described embodiments.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1 Design of Antisense Oligonucleotides Specific for a NucleicAcid Molecule Antisense to a Tumor Protein 63 (p63) and/or a SenseStrand of p63 Polynucleotide

As indicated above the term “oligonucleotide specific for” or“oligonucleotide targets” refers to an oligonucleotide having a sequence(i) capable of forming a stable complex with a portion of the targetedgene, or (ii) capable of forming a stable duplex with a portion of anmRNA transcript of the targeted gene.

Selection of appropriate oligonucleotides is facilitated by usingcomputer programs that automatically align nucleic acid sequences andindicate regions of identity or homology. Such programs are used tocompare nucleic acid sequences obtained, for example, by searchingdatabases such as GenBank or by sequencing PCR products. Comparison ofnucleic acid sequences from a range of species allows the selection ofnucleic acid sequences that display an appropriate degree of identitybetween species. In the case of genes that have not been sequenced.Southern blots are performed to allow a determination of the degree ofidentity between genes in target species and other species. Byperforming Southern blots at varying degrees of stringency, as is wellknown in the art, it is possible to obtain an approximate measure ofidentity. These procedures allow the selection of oligonucleotides thatexhibit a high degree of complementarity to target nucleic acidsequences in a subject to be controlled and a lower degree ofcomplementarity to corresponding nucleic acid sequences in otherspecies. One skilled in the art will realize that there is considerablelatitude in selecting appropriate regions of genes for use in thepresent invention.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo assays ortherapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays

The hybridization properties of the oligonucleotides described hereincan be determined by one or more in vitro assays as known in the art.For example, the properties of the oligonucleotides described herein canbe obtained by determination of binding strength between the targetnatural antisense and a potential drug molecules using melting curveassay.

The binding strength between the target natural antisense and apotential drug molecule (Molecule) can be estimated using any of theestablished methods of measuring the strength of intermolecularinteractions, for example, a melting curve assay.

Melting curve assay determines the temperature at which a rapidtransition from double-stranded to single-stranded conformation occursfor the natural antisense/Molecule complex. This temperature is widelyaccepted as a reliable measure of the interaction strength between thetwo molecules.

A melting curve assay can be performed using a cDNA copy of the actualnatural antisense RNA molecule or a synthetic DNA or RNA nucleotidecorresponding to the binding site of the Molecule. Multiple kitscontaining all necessary reagents to perform this assay are available(e.g. Applied Biosystems Inc. MeltDoctor kit). These kits include asuitable buffer solution containing one of the double strand DNA (dsDNA)binding dyes (such as ABI HRM dyes, SYBR Green, SYTO, etc.). Theproperties of the dsDNA dyes are such that they emit almost nofluorescence in free form, but are highly fluorescent when bound todsDNA.

To perform the assay the cDNA or a corresponding oligonucleotide aremixed with Molecule in concentrations defined by the particularmanufacturer's protocols. The mixture is heated to 95° C. to dissociateall pre-formed dsDNA complexes, then slowly cooled to room temperatureor other lower temperature defined by the kit manufacturer to allow theDNA molecules to anneal. The newly formed complexes are then slowlyheated to 95° C. with simultaneous continuous collection of data on theamount of fluorescence that is produced by the reaction. Thefluorescence intensity is inversely proportional to the amounts of dsDNApresent in the reaction. The data can be collected using a real time PCRinstrument compatible with the kit (e.g. ABI's StepOne Plus Real TimePCR System or lightTyper instrument, Roche Diagnostics, Lewes, UK).

Melting peaks are constructed by plotting the negative derivative offluorescence with respect to temperature (−d(Fluorescence)/dT) on they-axis) against temperature (x-axis) using appropriate software (forexample lightTyper (Roche) or SDS Dissociation Curve, ABI). The data isanalyzed to identify the temperature of the rapid transition from dsDNAcomplex to single strand molecules. This temperature is called Tm and isdirectly proportional to the strength of interaction between the twomolecules. Typically, Tm will exceed 40° C.

Example 2 Modulation of p63 Polynucleotide

Treatment of HEPG2 Cells with Antisense Oligonucleotide

HepG2 cells from ATCC (cat#HB-8065) were grown in growth media (MEM/EBSS(Hyclone cat #SH30024, or Mediatech cat #MT-10-010-CV)+10% FBS(Mediatech cat#MT35-011-CV)+penicillin/streptomycin (Mediatechcat#MT30-002-CI)) at 37° C. and 5% CO₂. On the day of the experiment themedia in the 6 well plates was changed to fresh growth media. Allantisense oligonucleotides were diluted to the concentration of 20 μM.Two μl of this solution was incubated with 400 μl of Opti-MEM media(Gibco cat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogencat#11668019) at room temperature for 20 min and applied to each well ofthe 6 well plates with HEPG2 cells. A Similar mixture including 2 μl ofwater instead of the oligonucleotide solution was used for themock-transfected controls. After 3-18 h of incubation at 37° C. and 5%CO₂ the media was changed to fresh growth media. 48 h after addition ofantisense oligonucleotides the media was removed and RNA was extractedfrom the cells using SV Total RNA Isolation System from Promega (cat #Z3105) or RNeasy Total RNA Isolation kit from Qiagen (cat#74181)following the manufacturers' instructions, 600 ng of RNA was added tothe reverse transcription reaction performed using Verso cDNA kit fromThermo Scientific (cat#ABI453B) or High Capacity cDNA ReverseTranscription Kit (cat#4368813) as described in the manufacturer'sprotocol. The cDNA from this reverse transcription reaction was used tomonitor gene expression by real time PCR using ABI Taqman GeneExpression Mix (cat#4369510) and primers/probes designed by ABI (AppliedBiosystems Taqman Gene Expression Assay: Hs00978340_ml by AppliedBiosystems Inc., Foster City Calif.). The following PCR cycle was used:50° C. for 2 min, 95° C. for 10 min, 40 cycles of (95° C. for 15seconds, 60° C. for 1 min) using Mx4000 thermal cycler (Stratagene).Fold change in gene expression after treatment with antisenseoligonucleotides was calculated based on the difference in18S-normalized dCt values between treated and mock-transfected samples.

Results: Real time PCR results show that the levels of p63 mRNA aresignificantly increased in HepG2 cells 48 h after treatment with oneantisense oligonucleotide to p63 natural antisense Hs.553263 (FIG. 1).

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

What is claimed is:
 1. A synthetic, modified oligonucleotide of 10 to 30 nucleotides in length comprising at least one modification wherein the at least one modification is selected from: at least one modified sugar moiety; at least one modified intemucleotide linkage; at least one modified nucleotide, and combinations thereof; wherein said oligonucleotide is an antisense compound which specifically hybridizes and is at least 95% complementary to a complementary 10 to 30 nucleotide region on a natural antisense polynucleotide of the p63 gene, wherein said natural antisense polynucleotide is selected from SEQ ID NO: 2 and upregulates the function and/or expression of a Tumor Protein 63 (p63) gene in vivo or in vitro as compared to a normal control.
 2. The oligonucleotide of claim 1, wherein the at least one modification comprises an intemucleotide linkage selected from the group consisting of: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.
 3. The oligonucleotide of claim 1, wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage.
 4. The oligonucleotide of claim 1, wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages.
 5. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide, said modified nucleotide selected from: a peptide nucleic acid, a locked nucleic acid (LNA), analogue, derivative, and a combination thereof.
 6. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof.
 7. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination thereof.
 8. The oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one modified sugar moiety selected from: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.
 9. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof.
 10. The oligonucleotide of claim 1, wherein the oligonucleotide comprises the sequences set forth as SEQ ID NO:
 5. 11. A composition comprising one or more oligonucleotides according to claim 1 and a pharmaceutically acceptable excipient.
 12. The composition of claim 11, wherein the oligonucleotides have at least about 95% sequence identity as compared to the nucleotide sequences set forth as SEQ ID NO:
 5. 13. The composition of claim 11, wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NO:
 5. 14. The composition of claim 13, wherein the oligonucleotides set forth as SEQ ID NO: 5 comprise one or more modifications wherein the one or more modifications are selected from: phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof.
 15. The composition of claim 14, wherein the one or more modifications are selected from: phosphorothioate, locked nucleic acid (LNA) molecules, and combinations thereof. 